V5-76-005
  PERSISTENCE AND DEGRADABILITY TESTING OF
BENZIDINE AND OTHER CARCINOGENIC COMPOUNDS
                     June 1976
                   FINAL REPORT

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This document is available to the public through the
       National Technical Information Service
            Springfield, Virginia  22151

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EPA 560/5-76-005                                                    TR 76-571
                  PERSISTENCE AND DEGRADABILITY TESTING OF
                 BENZIDINE AND OTHER CARCINOGENIC COMPOUNDS
                                   Authors

                              Philip H.  Howard
                              Jitendra Saxena
                       Contract No. 68-01-2679 Task 2
                                  June 1976
                               Project Officer

                               Carter Schuth
                                Prepared for

                         Office of Toxic Substances
                    U.S. Environmental Protection Agency
                           Washington, D.C. 20460

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This draft report has been reviewed by the Office
of Toxic Substances, EPA, and approved for publi-
cation.  Approval does not signify that the con-
tents necessarily reflect the views and policies
of the Environmental Protection Agency, nor does
mention of trade names or commercial products
constitute endorsement or recommendation for use.
                       ii

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                              TABLE OF CONTENTS
                                                                         Page
ABSTRACT                                                                   v

I.   Introduction                                                          1

II.  Review of the Analytical Method Used in SOCMA Studies                 2

III. Summary and Evaluation of Technical Information Developed on          6
     Environmental Degradation of Benzidine

     A.   Die-Away Tests in Chlorinated, Aerated, and                      6
          Non-Aerated Lake Water

     B.   Photolytic Studies in Deionized Aerated Water                    9

     C.   Biological Degradation - Activated Sludge System                13

     D.   Benzidine Ambient Monitoring Data                               15

     E.   Comparison of SOCMA Conclusions and Conclusions Supported       16
          by Research Data

IV.  General Discussion of Environmental Degradation Testing as           18
     Applied to Aromatic Amines and Carcinogens

V.   Conclusion                                                   '        21

REFERENCES                                                                22
                                     iii

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                               LIST OF TABLES


Number                                                                     Page;

  1     Benzidine Degradation Under Various Lake Water Conditions             7

  2     Benzidine Degradation in Chlorinated and Aerated Lake Water          8

  3     Photolysis Study of 100 ppb Benzidine in Deionized,  Aerated Water   11

  4     Photolysis of Benzidine at Various Concentrations in Deionized,     12
        Aerated Water Initially at 6.9 - 7.0 pH
                                      iv

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                                   ABSTRACT







     This report reviews and evaluates information on the environmental de-



gradation of benzidine which was generated by the Synthetic Organic Chemical



Manufacturer's Association (SOCMA) Benzidine Task Force and submitted to the



U.S. Environmental Protection Agency.   The experimental design,  execution,



and interpretation of the studies have been reviewed and evaluated.  It is



concluded that the SOCMA information is not sufficient for making intelligent


                                                                      14
regulatory decisions, and it is recommended that a test program  using   C-



benzidine be undertaken in order to generate the necessary information.

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I.    INTRODUCTION




     The U.S.  Environmental Protection Agency has  been working on promulgating




the Toxic Pollutant Effluent Standard for benzidine under Section 307(a)  of  the




Federal Water Pollution Control Act Amendments of  1972.   At the same time,  the




Office of Toxic Substances has been developing its capability to implement  the




concepts embodied in the Toxic Substances Control  Act regarding the need for




increased environmental testing of industrial chemicals.   Understanding the




fate of benzidine is, thus, important not only to  the development of an effec-




tive effluent standard but also as an example of voluntary industrial testing




for environmental effects.  Benzidine, besides being an important comnercial




chemical, is well-known to be carcinogenic:  incidence of bladder tumors among




workers exposed to benzidine is high, and the compound is reported to induce




hepatic tumors in mice and intestinal tumors in rats and to accelerate the




appearance of breast cancer in female rats.




     A review of the available literature  (Radding et al., 1975) conducted for




the Office of Toxic Substances, EPA, revealed that very little was known about




the environmental fate of benzidine.  In an effort to fill this information




gap, the Synthetic Organic Chemical Manufacturer's Association (SOCMA) Benzi-




dine Task Force has been working since 1973 to generate information necessary




for the development of the benzidine standard.  The SOCMA Benzidine Task Force




has made three submissions to EPA which have included information on benzidine




degradation.  The purpose of this report is to review the experimental design,




execution, and interpretation of these SOCMA submissions.  EPA's interests in




the analysis of the information include:   (1) regulatory decisions to be made




on benzidine; (2) the environmental fate of aromatic amines as a class; and




(3) the adequacy of the benzidine testing program as a prototype for environ-




mental fate testing.  These interests will also be addressed in this report.

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II.  REVIEW OF THE ANALYTICAL METHOD USED IN THE SOCMA STUDIES

     Analytical methods used to study environmental degradation of a chemical

can have considerable impact on the interpretation of the results.  In all the

SOCMA studies (SOCMA, 1975 a, b) a Chloramine-T colorimetric method was used.

The method has been proposed by EPA as an acceptable method for monitoring

benzidine and its salts in wastewater.  When used with water samples, the method

consists of the following steps:

     1.  Make sample basic (benzidine hydrochloride converted to undissociated
         benzidine).

     2.  Extract benzidine into ethyl acetate.

     3.  Extract benzidine from ethyl acetate with IN hydrochloric acid.

     4.  Add Chloramine-T (Sodium toluene-p-sulfonchloroamide)  to oxidize
         benzidine to a yellow meriquinoid complex.
H2N-
~Na++ HC1
                                                      HN =
                                                             NH
H2N<5>
\__/
/O^NH2
                                   2HC1
     5.
             HN =(    ) =\    > =NH
                  \/  \=/

             Meriquinoid Complex
Extract yellow meriquinoid complex with ethyl acetate (sometimes
chloroform) and record the absorption spectrum from 510 nm to 370 nm
(benzidine meriquinoid complex has a maximum absorbance at 436 nm)
for quantitative purposes.

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     This method appears to be extremely sensitive and specific for benzidine


and its salts in water.  The U.S.  EPA (1974) stated that the detection limit


was 0.2 ]ig/H, (ppb) when analyzing 1 liter samples.  Allied Chemical (Puliafico,


1975) has had some experience applying the method to wastewaters and they con-


cluded that the limit .of detection is not more than 0.5 ppb at 50% background

transmission with a 5 cm cell.  When the background is greater than 50% (com-


monly the case with dye manufacturing plant waste), the limit of detection


may be greater than 5 ppb.  When relatively uncontaminated water is used, a


low detection limit of 0.02 yg/fc (0.02 ppb) was reported by the Great Lakes

Laboratory (SOCMA, 1975 a, b).


     Besides being sensitive, the Chloramine-T colorimetric method is also

fairly specific for benzidine.  Any interferences would have to be extrac-
                                                                  \
ted into ethyl acetate from basic solution, extracted back into hydrochloric


acid, and develop a colored product on treatment with Chloramine-T that could


be extracted back into ethyl  acetate.  Analogs of benzidine, such as dichloro-


benzidine, -tolidine, and dianisidine (Classman and Meigs, 1951) can interfere,


but compounds such as  2,4-diaminobiphenyl, aniline, a- and g-naphthylamine


give no  color on Chloramine-T treatment and, therefore, do not interfere (Butt

and Strafford, 1956).  Recording the visible spectrum of the complex also adds


an extra degree of specificity, since the  interfering analogs have different


maximum  absorptions.

     Thus, overall, the analytical method  used in the SOCMA submissions is a

very sensitive and specific method for only benzidine or its salts.  This con-

clusion  in itself is an important one which impacts on the interpretation of


all of the results.  Because  of the selected analytical method, or any method


that measures only the parent compound, all the measurements of benzidine

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degradation are really only measurements of the loss  of  benzidine.   Very  slight




alterations may result in no detection by the analytical method used.   For




example, Sciarini and Meigs (1958)  noted that although the Chloramine-T method




is very sensitive for benzidine,  it is "much less sensitive for 3-monohydroxy-




benzidine, 3,3'-dihydroxybenzidine, and -aminophenol."   In fact,  dihydroxy-




benzidine added to urine could not  be extracted with any solvent.




     With benzidine, slight chemical changes might be quite significant.   It




is postulated that carcinogenic aromatic amines must undergo metabolic activa-




tion to a proximate carcinogen prior to tumor induction.  The following ring-




and N-hydroxylated products are regarded as the likely proximate carcinogens




of benzidine (Gadian, 1975; Arcos and Argus, 1975):
       3-hydroxybenzidine
       3,3'-dihydroxybenzidine
       N-hydroxybenzidine
       N, N'-dihydroxybenzidine
                                             H2N-
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     These compounds might be formed from benzidine,  but would not be detected




by the Chloramine-T method.  Thus,  although the Chloramine-T method would measure




the loss of benzidine, that loss may not correspond to a total degradation of




its likely proximate carcinogens.

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III.   SUMMARY AND EVALUATION OF TECHNICAL INFORMATION  DEVELOPED  ON  ENVIRONMENTAL
      DEGRADATION OF BENZIDINE

     A.    Die-Away Tests in Chlorinated,  Aerated,  and  Non-Aerated Lake Water-
          Experimental Design, Results,  and Evaluation

          Two experiments on the degradation of benzidine in lake water were

conducted.  The first (SOCMA, 1975 a)  consisted of measuring the loss  of benzi-

dine in lake water (obtained from a Buffalo, NY pumping station) which was:

(1) maintained at 1 mg/SL available chlorine, (2) maintained at 2 mg/  available

chlorine, (3) stirred, (4) aerated, and (5) undisturbed.   Initial benzidine

concentrations of 1, 2, and 5 yg/ were used and all colorimetric analyses

were performed in triplicate.  The investigators indicated that all attempts

were made to shield the experimental set-up from light.  The results are pre-

sented in Table 1.

          In the second study on benzidine degradation in water, three condi-

tions were used:  (1) lake water brought to an initial chlorine concentration

of 1 ppm, (2) lake water brought to an initial chlorine concentration of 2 ppm,

and (3) vigorously aerated lake water (approximately 10 H of air per hour).

The initial benzidine concentration ranged between 1 to 70 ppb.  Benzidine and

dissolved oxygen levels were measured at various intervals.  Table  2 summarizes

the results.  The investigators subjected the data to statistical analysis and

concluded that the data showed no significant differences under the different

conditions.  By plotting the reaction rates, the investigators concluded that

the reaction was probably 1st order with a rate constant of 0.175 per hour

(half-life = 4 hours).

          These results indicate rather conclusively that something is happening

at a relatively fast rate to benzidine that is placed in lake water.  However,

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Table  1.   Benzidine  Degradation Under Various Lake Water Conditions (SOCMA,  1975 a)
                                             Benzldlne Concentration  (pg/8.)
Conditions
Chlorinated Lake Water
(1 mg/e C12)




Chlorinated Lake Water
(2 mg/i C12)




Aerated Lake Water





Stirred Lake Water





Undisturbed
Lake Water





Time Solution A
Oirs.) (initial ^ 1 ue/)O
0.5
4
12
24
48
72
0.5
4
12
24
48
72
0.5
4
12
24
48
72
0.5
4
12
24
48
72
0.5
4
12
24
48
72
0.84
0.48
0.10
N.D.
-

0.79
0.39
0.04
N.D.
-
-
0.83
0.69
0.31
N.D.
-

0.90
0.78
0.48
N.D.
-
-
0.89
0.71
0.47
0.02
N.D.

Solution B
(Initial ^ 2 ufc/8.)
1.90
1.17
0.68
N.D.
.
-
1.73
1.09
0.31
N.D.
-'
-
l.')4
l.Ai.
0.7.J
N.J).
-
-
1.8:
l..")7
0.80
N.n.
-
-
1.85
1.45
0.76
N.D.
-
-
Solution C
(initial - 5 ue/O
4.42
4.06
2.17
N.D.
-
-
4.37
4.10
1.87
N.D.
-
-
4.61
4.27
2.01
N.D.
-
-
4.70
4.21
2.15
N.D.
-
-
4.8J
4.03
2.07
N.D.
-
-
             N.D. = Not detectable (<0.02 wg/4)

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                Table 2.   Benzidine Degradation in  Chlorinated and Aerated Lake Water  (SOCMA, 1975 b).
                                                          Benaidine Concentration
oo
Conditions
Chlorinated
Lake Water
1 ppm C12


Chlorinated
Lake Water
2 ppm C12


Aerated
Lake Water



Time
(hrs.)
0.5
3
6
12
24
0.5
3
6
12
24
0.5
3
6
12
24
Initial 1 *
Benzidine 0
0.89
0.51
0.39
0.09
<0.02
0.92
0.49
0.28
0.10
<0.02
0.87
0.58
0.40
0.28
<0.02
92.3
89.2
87.3
88.0
85.6
91.7
89.7
88.6
87.2
88.0
100
100
100
100
100
Initial 5 * Initial 10 A
Benzidine 0. Benzidine 0
4.47
3.97
2.93
2.03
<0.02
4.81
3.87
3.01
1.83
<0.02




91.6
87.6
87.3
88.2
86.3
92.3
87.8
86.9
85.4
86.7




9.70
6.81
5.37
4.18
0.73
9.64
6.95
5.08
3.73
0.52
9.37
8.02
5.43
4.00
0.13
93.0
89.3
88.1
87,9
88.0
96.4
88.3
88.0
87.3
86.9
100
100
100
100
100
Initial 50 *
Benzidine C<
47.36
36.21
28.34
18.69
1.05
46.73
34.77
29.32
17.97
1.21




94.7
87.4
88.3
86.9
85.8
94.4
89.1
88.3
86.9
87.7




Initial 70 ^
Benzidine 0
68.33
42.76
32.81
21.84
1.62
67.24
43.81
30.96
21.32
1.19
66.90
43.51
32.84
25.00
1.46
93.2
88.1
87.8
88.1
88.0
93.2
88.0
89.2
87.6
88.0
100
100
100
100
100
                          Dissolved oxygen  (% saturation)

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as previously discussed, the analytical method allows  no  insight  into  the

degree or pathway of degradation.   After 48 hours,  no  benzidine is  detectable

by the colorimetric method,  even at relatively high starting concentrations.

Unfortunately, little insight into the mechanism of decay is provided  by the

available work.  By protecting the reaction vessels from light, photolysis

seems to be ruled out as a mechanism, although, as  will be discussed later,

benzidine may be sensitive to light.  Microbial degradation is probably not

very important in the first degradation step of benzidine since chlorinated

water has a rate similar to unchlorinated water and the rate of benzidine  dis-

appearance is extremely fast.  The use of a sterilized sample would be neces-

sary to demonstrate conclusively a non-microbial mechanism.

          The data are insufficient to demonstrate  an  air oxidation mechanism.

Such a mechanism would have been strongly implied if no benzidine disappearance

occurred in a deoxygenated lake water sample, but no such sample  was run.   Also,

the available information does not indicate that chlorination is  an important

degradation mechanism, since the reaction rates are similar in chlorinated and

unchlorinated water.  It is unfortunate that the pH of the lake water was ap-

parently not recorded, since it is quite possible that the reaction may be pH

dependent.  Also, knowing the pH would have clarified whether volatilization

was an important loss mechanism, especially in aerated runs.  (Under acid con-

ditions the benzidine would be present as a non-volatile salt.)

     B.   Photolysis Studies in Deionized, Areated Water - Experimental Design,
          Results, and Evaluation

          Two series of studies on benzidine photolysis in deionized water were

conducted (SOCMA, 1975 a, b).  The first study CSOCMA, 1975 a) consisted of

irradiation of a 100 ppb solution of benzidine in deionized water.   The 100 ppb

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benzidine solution was made from dilution of a 1000 ppm benzidine stock solu-




tion (added as dihydrochloride) with aerated deionized water.   The solution




was placed in a 250 mfc quartz, flat bottom flask and set in a Fade-Ometer




irradiation apparatus (Atlas Electric Devices, Co., Fade-Ometer,  Model 25-FR)




fitted with a xenon lamp.  The xenon lamp provides light irradiation compara-




ble to sunlight in the important ultraviolet region C300 - 450 nm).  Twenty-




three hours of Fade-Ometer exposure are approximately equal to 20 hours of




June sunlight, when the standardization is based on the bleaching of a stan-




dard dye cloth (Reiter, 1975).  Analysis of benzidine at various  irradiation




times was accomplished using a modified Chloramine-T colorimetric method.  The




water sample was acidified, Chloramine-T was added, and the colored meriquinoid




complex was extracted with chloroform for quantitation.  The clean-up procedure




and ethyl acetate extraction described previously were not used.   Analysis was




performed on 100 mfc of solution and the detection limit was 5 ppb.  The results




are presented in Table 3.  For each time period, a complete flask was sacrificed




for analysis (in most cases duplicate samples were tested) in order to avoid




the effect of level changes.  The reaction mixture was initially at room tem-




perature and after 2.5 and 24 hours of irradiation was at 37 - 38C.  The pH




was not reported.




          The second study (SOCMA, 1975 b) used a wider variety of concentra-




tions, increased the sensitivity of the colorimetric method (limit of detec-




tion 0.1 ppb), and adjusted the pH before irradiation to 6.9 - 7.0 with sodium




hydroxide.  The temperature was at 45 - 47C which was significantly different




from the first study.  The results are presented in Table 4.




          Because of the experimental design of these studies, it is very dif-




ficult to determine the importance of photochemical degradation in the environment.
                                    10

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 Table  3.  Photolysis Study of 100 ppb Benzidine in Deionized, Aerated Water
           (SOCMA,  1975 a)
Time **
(hrs.)
0
0.5
0.5
1.5
1.5
2.5
2.5
4.0
4.0 .
12.0
12.0
24
Ug in Sample of 100 mfc
Test 1 Test 2
10
7.86
6.99
2.02
 *
1.63
2.22




<0.5
10.0
3.38
5.63
1.47
1.80


0.45
0.177
N.D.
0.00639
N.D.
% of initial Concentration
Test 1 Test 2
100
78.6
69.9
20.2
 *
16.3
22.2


<5


100
33.8
56.3
14.7
18.0


0.45
1.73

0.064

 * Sample obviously degraded
** Samples with identical
   times were duplicates
    Temperature

 Room Temperature
Initially, 37-38C at
    Equilibrium
                                      11
                                                                                         J

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Table 4.   Photolysis of Benzidine at Various  Concentrations  in  Deionized, Aerated
          Water Initially at 6.9 - 7.0 pH (SOCMA,  1975  b)
Time
0
0.5
1.5
4.0
12.0
24.0
27.0
28.0
29.0
48.0
53.0
56.0
72.0
77.0
80.0



1.0
0.73
0.66
0.45
0.087
< 0.002
< 0.001
-
-
-
-
-
-
-
-
Temperature
Room temperature
initially, 41C in
1.5 hrs., remained
at 46-47C for the
remainder of the
times
ppra Benzidine
10
8.70
8.05
6.45
4.65
2.49
-
-
1.80
0.50
0.29
-
0.018
0.018
-
Temperature
45-49C after
first 4 hours.


100
91.95
86.93
95.61*
75.41
63.68
-
52.13
-
-
-
31.15
-
-
22.92
Temperature
Varied from 47C
after 1.5 hrs. to
55C.

   * Irregularity in the 4 hr. value may have been  caused  by  adsorption on the
     solids which participated at the high concentrations.
                                     12

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Since aerated, deionized water was used, it is possible that  all  the  loss  of




benzidine could be due to the chemical degradation noted in the previous section.




Apparently, no non-irradiated or deoxygenated water controls  were prepared for




comparison.  The elevated temperatures encountered in samples in  the  fadeometer




could have accelerated non-photolytic and photolytic breakdown.  The  rates of




benzidine lost in aerated lake water samples are not that different from the




losses noted in the irradiated samples.  Controls consisting  of an aerated,




non-irradiated sample maintained at temperatures comparable to the irradiated




samples as well as a deoxygenated, irradiated sample, would have  been useful.




Varying the pH would also have facilitated interpretation, since  the  ultraviolet




absorption spectra is pH-dependent.  The hydrogen ion concentration may also




affect the efficiency and mechanism of the photochemical process  if photodegra-




dation Is taking place.  Furthermore, it has been shown that  photolysis in




natural waters may be substantially different than photochemical  processes in




purified water (e.g., Zepp et^ ai., 1975); thus, photolysis in natural water




(perhaps lake water) would also have been useful.




     C.   Biological Degradation - Activated Sludge System




          Biodegradation is frequently the most important process for degrading




a chemical contaminant in the environment.  Very little information is available




on the biodegradability of benzidine.  Lutin and coworkers (1965) studied the




biodegradation of benzidine using a Warburg apparatus, unacclimated activated




sludge C2,500 mg/Jl suspended solids), and a benzidine concentration of 500 mg/.




They found that the oxygen uptake was less than the control,  suggesting that




benzidine was not biodegraded and/or the growth of the degrading microorganisms




was inhibited by the presence of benzidine.






                                    13

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          The SOCMA Benzidine Task Force also  has  examined  the biological




degradation of benzidine.   In one study (SOCMA,  1975  a) benzidine was added




at a concentration of 110 mg/fc to a model activated sludge  treatment plant.




The study indicated that the benzidine remained  substantially unchanged  in




the system effluent and had no adverse effect  on the  normal BOD  reduction




of the system.  In a later study (SOCMA, 1975  b),  the activated  sludge pilot




system was first acclimated to aniline at 50 tng/X  and then  fed 40 ppb of




benzidine (aniline remained at 50 ppm).  A continuous week  of feeding benzidine




at approximately 45 yg/ had little effect on  BOD-COD reduction, indicating




no toxic effects of benzidine to the microorganisms.   The liquid effluent




and sludge phase were analyzed by the Chloramine-T colorimetric  method in




order to provide a mass-balance of benzidine.   Of the 1579.5 yg  of  benzidine




fed during the week run, 202.5 yg were recovered in the liquid effluent  or




sludge phase.  The investigators concluded that  1377  yg had been biologically




degraded or oxidized.




          In the third study  (SOCMA, 1975 c) a variety of conditions  and effects




were studied.  Warburg tests using benzidine as the test substrate  were  run




with both acclimated and non-acclimated sludge (2000  - 2500 mg/ Mixed Liquor




Suspended Solids).  The rate of oxygen uptake was compared  to the  control to




determine if the benzidine was toxic to the microorganisms.  An  inhibitory




effect was noted somewhere between 40 mg/ to 80 mg/JZ, of benzidine  for the




unacclimated sludge and between 60 mg/Jl to 120 mg/ of benzidine for  the ac-




climated sludge.  In the first few hours the oxygen consumed was 2% of the




theoretical oxygen demand (amount of oxygen required  to convert  benzidine  to




carbon dioxide and water) for the unacclimated sludge, and  2.8%  for the  ac-




climated sludge.  The activated sludge pilot system described above was  also





                                    14

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run for six weeks after acclimation with 50 yg/  of benzidine and no aniline.




During the six week period 14,168 yg of benzidine were fed to the system,  of




which 13,506 (almost 95%) were oxidized.  Adsorption to the sludge was considered.




A sterile control was run in order to determine the amount of oxidation attribu-




table to air oxidation.  It was concluded that approximately 30% of the oxida-




tion could be assigned to air oxidation.  Also, an unacclimated activated sludge




run was also undertaken which demonstrated approximately the same benzidine




reduction (.95%) as the acclimated test.




          The above results are difficult to interpret in terms of biodegrada-




tion in nature.  The microbial concentrations and communities, and nutrient




concentrations of an activated sludge treatment plant resemble secondary sewage




treatment plant conditions but are very different from natural environmental




conditions.  The results from the Warburg test, which simulates activated




sludge treatment conditions, suggest some breakdown, but oxygen consumption




equivalent to 2 - 2.8% of theoretical oxygen demand is not suggestive of an




easily biodegraded compound.  However,  the pilot plant studies do indicate




that sizable portions of benzidine in a waste stream can be removed by a com-




bination of air oxidation and bio-oxidation.  Whether similar oxidation in




natural waters will occur is still unknown.




     D.   Benzidine Ambient Monitoring  Data




          Ambient monitoring data can often provide insight into the persistence




of a chemical contaminant in the environment.  Detection of a chemical usually




provides clear evidence of some resistance to degradation.  However, lack of




detection is somewhat harder to interpret because there may be several reasons




for the lack of detection besides degradation  (e.g., poor analytical method,




strong adsorption of the material onto  soil or sediment).
                                    15

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          The SOCMA Task Force (SOCMA, 1975 b)  conducted a field survey to

analyze water and sediment samples for benzidine in the Buffalo River Watershed

upstream and downstream from Allied Chemical Corporation's Specialty Chemical

Division Plant at Buffalo.  It was believed that benzidine was being discharged

from this plant.

          During July 1, 2, and 3, 1975, 21 sediment and 42 water samples were

gathered from 7 sites.  Each sample was maintained in ice-water until it arrived

at the analysis laboratory (less than 20 minutes).  Analysis using the Chlora-

mine-T method indicated that the concentrations of benzidine in the sediment

or water samples were below the level of detectability (~ 0.2 pg/Jl when using

1 liter samples).  A similar study of water samples from the Niagara River and

near the intake for the Tonawanda Water Treatment Plant showed no detectable

benzidine.

          These results suggest that benzidine may be altered in the aqueous

environment to the extent that it is no longer detectable by the colorimetric

method.  However, the lack of detection could also be due to dilution below

the limit of detection.  Interpretation of the results in terms of degrada-

bility would have been much clearer if the amounts of benzidine emitted into

the water systems were available as well as a calculated maximum concentration

estimated from dilution due to the flow of the rivers.  It should be kept in

mind that benzidine has been detected in the Sumida River in Japan (Takemura

et'al., 1965).

     E.   Comparison of SOCMA Conclusions and Conclusions Supported by Research
          Data

          From the studies discussed above, the SOCMA Benzidine Task Force

(SOCMA, 1975 a) has reached the conclusion that "benzidine is destroyed in

                                   16

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nature by air oxidation, biological oxidation,  and sunlight."   In the  second




submission, SOCMA (1975 b) notes in the summary that "it has been shown that




benzidine is not a persistent compound.  Laboratory data have  indicated that




benzidine is readily destroyed by naturally occurring processes [emphasis




added by SOCMA]."  In the final submission, SOCMA (1975 c)  concludes that




"benzidine is not a persistent chemical but is destroyed by bio-oxidation,




air oxidation, exposure to ultraviolet light and chlorination  as practiced




in water treatment."  In order to understand whether these conclusions are




justified by the experimental data, such terms as "persistence" and "destroyed"




must be defined.  The term "destroyed" implies to us that the  chemical is




totally broken up or degraded to products such as carbon dioxide, water, and




nitrogen oxides (usually referred to as mineralization).  The  term "persistence"




has had many interpretations (see Howard et al., 1975), but we prefer to define




non-persistent compounds as chemicals that are degraded in a relatively short




period of time to naturally occurring low molecular weight metabolites or that




are mineralized.  However, non-persistent chemicals are not compounds that




undergo slight chemical alterations, which is all that can be measured with the




Chloramine-T method.  In the detergents industry slight chemical alterations




of detergents have been referred to as "primary degradation" (Swisher, 1970).




          Thus, the available information does not support the statement that




benzidine is readily "destroyed" by naturally occurring processes.  Of the




three mechanisms of degradation  (air oxidation, biological oxidation, or sun-




light) , only air oxidation has convincingly been shown to have such an effect.




The lack of controls in the photolysis studies and the poor simulation of nature




in the biological oxidation studies preclude any firm conclusions on these two




possible degradation mechanisms.  The biological oxidation studies do indicate,




however, that degradation of benzidine is likely to occur in a secondary sewage




treatment plant.




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IV.  GENERAL DISCUSSION OF ENVIRONMENTAL DEGRADATION  TESTING AS  APPLIED TO

     AROMATIC AMINES AND CARCINOGENS



     From the above discussion, it is our conclusion  that the information



available on the environmental persistence or degradability of benzidine  is



not sufficient to make an intelligent regulatory decision.  Benzidine is  a



carcinogen whose activity is thought to be due to some of its metabolites.



Without understanding the pathways and metabolites resulting from degradation



in the environment, the hazards associated with the presence of  benzidine in



the environment cannot truly be assessed.



     Making predictions concerning the environmental  persistence of aromatic



amines as a class from the data generated for benzidine would require some



understanding of the mechanism and pathways of degradation of benzidine.   Since



such information is not available in the SOCMA reports, meaningful predictions



cannot be made other than the suggestion that aromatic amines may be susceptible



to air oxidation.



     From the generally negative conclusions in this  report, it  is obvious



that we would not recommend this benzidine testing program as a prototype for



environmental fate testing, especially for carcinogenic chemicals.  This  is



indeed unfortunate since the money and effort spent on the benzidine work,



with very slight modification, could have produced excellent results.



     Our major criticism of the benzidine protocol is the analytical method



used.  The analytical method is sensitive and specific, but it only measures


                     14
benzidine.  By using   C-labelled benzidine, which is readily available



($111/50 yCi, New England Nuclear, personal communication), the same or better



sensitivity could have been maintained, but a mass balance of benzidine and



breakdown products could have been developed.  Labelling the compound also



would have facilitated isolation of the metabolites.
                                     18

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     The reaction conditions used with benzidine are fairly  realistic  considering



that benzidine's major source of contamination is from water effluents.  However,



much better controls are necessary to distinguish between the mechanisms of  de-



gradation.  It is important to understand the mechanism of degradation in  order



to determine the variability of results in nature.   For example,  if  biodegrada-



tion is the major mechanism, the rates of degradation will vary considerably



depending upon the microbial concentration and population, carbon sources, accli-



mation, etc.  However, if air oxidation is the major degradation mechanism,  the



rates should be fairly constant in different microenvironments.


                                                             14
     We would recommend a testing program that would include   C-benzidine in



lake or river water, lake or river water with sediment, sterilized lake or



river water (with and without sediment), and distilled water.  The pH of the



water should be buffered to acidic, neutral, and basic conditions (pH 5-9) to



determine the effect of the hydrogen ion concentration.  Controls with and with-



out light should be run to determine the effect of laboratory light.  Photolysis



studies should be run with distilled water and simulated sunlight at tempera-



tures and pH's comparable to dark samples.  The effect of oxygen concentration



in irradiated and non-irradiated samples should be determined by deoxygenating



some controls (probably by bubbling nitrogen through the solution).   Sediment



should be included in at least one test because benzidine has been shown to



adsorb to some clays (Furukawa and Brindley, 1973; pH-dependent adsorption,



Lahav and Raziel, 1971 a, b), and this may alter the rate and pathway of de-



gradation.



     Attempts to isolate and identify metabolites will vary  depending upon


          14                            14
where the   C label resides.  Traps for   CO. and any volatile metabolites
                                     19

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should be included so that a mass balance  will be possible.  Emphasis should




be placed on trying to detect some of the  suspected  proximate  carcinogens of




benzidine.  The ring dihydroxyl derivative,  if it is formed, may be so water




soluble that it will be difficult to isolate.  The method  of Sternson (1975)




may be helpful in detecting hydroxylamine  degradation products.
                                      20

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V.   CONCLUSION



     Information developed by the SOCMA Benzidine Task Force suggests  that



benzidine in natural water may be rapidly air oxidized to the extent that it



is no longer detected by a Chloramine-T colorimetric method.   There is no evi-



dence, however, that the loss of color using Chloramine-T method corresponds



to a loss of carcinogenic activity.  The possibility of biological oxidation



or photodegradation of benzidine taking place in aqueous systems cannot be



decided from the available information, although degradation under conditions



of secondary sewage treatment appears likely.  It is suggested that further


                          14
studies be conducted with   C-benzidine and attempts be made to determine  the



degradation pathways and metabolites and to distinguish between chemical, photo-



chemical, and biological degradation.
                                     21

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                                 REFERENCES


Arcos, J.C. and Argus, M.. (1974), "Structure-Activity Relationships," Chap.  5
     in Chemical Induction of Cancer - Structural Bases and Biological Mechanisms,
     Academic Press, New York.

Butt, L.T. and Strafford, N. (1956), "Papilloma of the Bladder in the Chemical
     Industry - Analytical Methods for the Determination of Benzidine and 2-
     Naphthylamine, Recommended by A.B.C.M. Sub-Committee," J. Appl. Chem.,
     j>, 525-39.

Furukawa, T. and Brindley, G.W. (1973), "Adsorption and Oxidation of Benzidine
     and Aniline by Montmorillonite and Hectrite", Clay and Clay Minerals,
     2, 279-88.

Gadian, T.  (1975), "Carcinogens in Industry, With Special Reference to Dichloro-
     benzidine," Chem. Indust., 19, 821-31, (Oct. 4).

Howard, P.H., Saxena, J., Durkin, P.R., and Ou, L.T. (1975), "Review and Evalua-
     tion of Available Techniques for Determining Persistence and Routes of
     Degradation of Chemical Substances in the Environment," U.S. Nat. Tech.
     Inform. Serv., PB 243 825-7WP, EPA-560/5-75-006.

Lahav, N. and Raziel, S.  (1971 a), "Interaction Between Montmorillonite and
     Benzidine in Aqueous Solutions. I. Adsorption of Benzidine on Montmorillo-
     nite," Isr. J. Chem., JK6), 683-9.

Lahav, N. and Raziel, S.  (1971 b), "Interaction Between Montmorillonite and
     Benzidine in Aqueous Solutions. II. General Kinetic Study," Isr. J. Chem.,
     2(6),  691-4.

Lutln, P.A., Cibulka, J.J., and MaIaney, G.W. (1965), "Oxidation of Selected
     Carcinogenic Compounds by Activated Sludge," Purdue Univ., Eng. Bull.,
     Ext. Ser. No. 118,  131-45.

Puliafico,  S.J.  (1975),  "Evaluation of Proposed EPA Method for Benzidine and
     Its Salts in Wastewaters," in SOCMA (1975 a).

Radding, S.B., Holt, B.R., Jones, J.L., Liu, D.H., Mill, T., and Hendry, D.G.
      (1975), "Review of  the Environmental Fate of Selected Chemicals," U.S.
     Nat. Tech. Inform.  Serv., PB 238-908, EPA 560/5-75-001.

Reiter, W.M. (1975), Personal  communication, December 10, Allied Chemical,
     Morristown, New Jersey.

Sciarini, L.J. and Meigs, J.W. (1958), "Biotransformation of Benzidine, an
     Industrial Carcinogen, in the Dog," A.M.A. Arch. Ind. Health, 18, 521-30.
                                      22

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SOCMA (1975 a),  "First Submission of the SOCMA Benzidine  Task  Force  to Environ-
     mental Protection Agency,"  June 5.

SOCMA (1975 b),  "Second Submission,  Status Report on Benzidine Control, Benzidine
     Task Force, Synthetic Organic Chemical Manufacturer's  Association to  the
     Environmental Protection Agency,"  August 5.

SOCMA (1975 c),  "Third Submission to the U.S. Environmental Protection Agency
     by the SOCMA Benzidine Task Force," November 12.

Sternson, L.A.  (1975)> "Detection of Arylhydroxylamines as  Intermediates  in
     the Metabolic Reduction of Nitro Compounds," Experimentia, 31X3), 268-70.

Swisher, R.D. (1970), Surfactant Biodegradation,  Marcell Dekker, Inc., New York.

Takemura, N., Akiama, T., and Nakajima,  C. (1965), "A Survey of the  Pollution
     of the Sumida River, Especially on the Aromatic Amines in the Water,"
     J. Air Water Pollution, JK10),  665-70.

U.S. EPA (1974), "Method for Benzidine and Its Salts in Wastewaters," in
     SOCMA  (L975 a).

Zepp, R.G., Wolfe, N.C., Gordon, J.A., and Baughman, G.L. (1975), "Dynamics of
     2,4-D Esters in Surface Waters. Hydrolysis,  Photolysis, and Vaporization,"
     Environ. Sci. Technol, 9(13), 1144-50.
                                     23

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                                   TECHNICAL REPORT DATA
                            (Please read laaructions on the reverse before completing)
1. REPORT NO.
                              2.
                                                            3. RECIPIENT'S ACCESSION-NO.
4. TITLE AND SUBTITLE
Persistence and Degradability  Testing of Benzidine
and Other Carcinogenic Compounds
             5. REPORT DATE
                June 1976
             6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)

Philip H.  Howard and Jitendra Saxena
             8. PERFORMING ORGANIZATION REPORT NO

                TR 76-571
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Center  for Chemical Hazard  Assessment
Syracuse Research Corporation
Merrill Lane
Syracuse, NY   13210
                                                            10. PROGRAM ELEMENT NO.
             11. CONTRACT/GRANT NO.


               EPA 68-01-2679
12. SPONSORING AGENCY NAME AND ADDRESS

Office  of Toxic Substances
U.S.  Environmental Protection Agency
Washington, D.C.   20460
             13. TYPE OF REPORT AND PERIOD COVERED
               Final Report	
             14. SPONSORING AGENCY CODE
15. SUPPLEMENTARY NOTES
16. ABSTRACT
      This report reviews  and evaluates information on the environmental degradation
of  benzidine that was  generated by the Synthetic Organic Chemical Manufacturer's
Association (SOCMA) Benzidine Task Force and  submitted to the U.S.  Environmental
Protection Agency.  The experimental design,  execution, and interpretation of the
studies have been reviewed and evaluated.   It is concluded that  the SOCMA information
is  not sufficient for  making intelligent regulatory decisions and a test program
using -^C-benzidine is recommended in order to generate the necessary information.
17.
                                KEY WORDS AND DOCUMENT ANALYSIS
                  DESCRIPTORS
          benzidine
          environmental fate testing
          persistence
                                               b.lDENTIFIEHS/OPEN ENDED TERMS
                           c.  COSATI l-'iclcl/Group
18. DISTRIBUTION STATEMENT
Document is available to public through  the
National Technical  Information Service,
Springfield, Virginia   22151	
19. SECURITY CLASS (This Report)
21. NO. OF PAGtS
     23
20. SECURITY CLASS (Thispage)
                           22. PRICE
EPA Form 2220-1 (9-73)

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